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Importance: Persistence of FLT3 internal tandem duplication (ITD) in adults with acute myeloid leukemia (AML) in first complete remission (CR) prior to allogeneic hematopoietic cell transplant (HCT) is associated with increased relapse and death after transplant, but the association between the level of measurable residual disease (MRD) detected and clinical outcome is unknown. Objective: To examine the association between pre-allogeneic HCT MRD level with relapse and death posttransplant in adults with AML in first CR. Design, Setting, and Participants: In this cohort study, DNA sequencing was performed on first CR blood from patients with FLT3-ITD AML transplanted from March 2013 to February 2019. Clinical follow-up was through May 2022. Data were analyzed from October 2022 to December 2023. Exposure: Centralized DNA sequencing for FLT3-ITD in pre-allogeneic HCT first CR blood using a commercially available kit. Main Outcomes and Measures: The primary outcomes were overall survival and cumulative incidence of relapse, with non-relapse-associated mortality as a competing risk post-allogeneic HCT. Kaplan-Meier estimations (log-rank tests), Cox proportional hazards models, and Fine-Gray models were used to estimate the end points. Results: Of 537 included patients with FLT3-ITD AML from the Pre-MEASURE study, 296 (55.1%) were female, and the median (IQR) age was 55.6 (42.9-64.1) years. Using the variant allele fraction (VAF) threshold of 0.01% or greater for MRD positivity, the results closely aligned with those previously reported. With no VAF threshold applied (VAF greater than 0%), 263 FLT3-ITD variants (median [range] VAF, 0.005% [0.0002%-44%]), and 177 patients (33.0%) with positive findings were identified. Multivariable analyses showed that residual FLT3-ITD was the variable most associated with relapse and overall survival, with a dose-dependent correlation. Patients receiving reduced-intensity conditioning without melphalan or nonmyeloablative conditioning had increased risk of relapse and death at any given level of MRD compared with those receiving reduced-intensity conditioning with melphalan or myeloablative conditioning. Conclusions and Relevance: This study provides generalizable and clinically applicable evidence that the detection of residual FLT3-ITD in the blood of adults in first CR from AML prior to allogeneic HCT is associated with an increased risk of relapse and death, particularly for those with a VAF of 0.01% or greater. While transplant conditioning intensification, an intervention not available to all, may help mitigate some of this risk, alternative approaches will be necessary for this high-risk population of patients who are underserved by the current standard of care.
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Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Neoplasia Residual , Trasplante Homólogo , Tirosina Quinasa 3 Similar a fms , Humanos , Tirosina Quinasa 3 Similar a fms/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/sangre , Femenino , Masculino , Persona de Mediana Edad , Trasplante de Células Madre Hematopoyéticas/métodos , Adulto , Secuencias Repetidas en Tándem , Anciano , Duplicación de Gen , Estudios de CohortesRESUMEN
The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials and discrepancies have been observed between different techniques for MRD assessment. In 62 patients with AML, aged 18-60 years, in first complete remission after intensive induction therapy on the randomized phase III SWOG-S0106 clinical trial (clinicaltrials gov. Identifier: NCT00085709), MRD detection by centralized, high-quality multiparametric flow cytometry was compared with a 29-gene panel utilizing duplex sequencing (DS), an ultrasensitive next-generation sequencing method that generates double-stranded consensus sequences to reduce false positive errors. MRD as defined by DS was observed in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs. 13%; hazard ratio [HR] =8.8; 95% confidence interval [CI]: 3.2-24.5; P<0.001) and decreased survival (32% vs. 82%; HR=5.6; 95% CI: 2.3-13.8; P<0.001) at 5 years. DS MRD strongly outperformed multiparametric flow cytometry MRD, which was observed in ten (16%) patients and marginally associated with higher rates of relapse (50% vs. 30%; HR=2.4; 95% CI: 0.9-6.7; P=0.087) and decreased survival (40% vs. 68%; HR=2.5; 95% CI: 1.0-6.3; P=0.059) at 5 years. Furthermore, the prognostic significance of DS MRD status at the time of remission for subsequent relapse was similar on both randomized arms of the trial. These findings suggest that next-generation sequencing-based AML MRD testing is a powerful tool that could be developed for use in patient management and for early anti-leukemic treatment assessment in clinical trials.
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Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Adulto , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Resultado del Tratamiento , Pronóstico , Recurrencia , Neoplasia Residual/diagnóstico , Citometría de Flujo/métodosRESUMEN
Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to comparable strategies for rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from samples with known SV properties, we demonstrate that duplex sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of newly created SVs, suggesting end joining as a possible formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.
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The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials. Discrepancies have been observed between different techniques for MRD assessment and there remains a need to compare centralized, high-quality multiparametric flow cytometry (MFC) and ultrasensitive next-generation sequencing (NGS) in AML patients with diverse mutational profiles. In 62 patients with AML, aged 18-60, in first complete remission after intensive induction therapy on the randomized phase 3 SWOG-S0106 clinical trial, MRD detection by MFC was compared with a 29 gene panel utilizing duplex sequencing (DS), an NGS method that generates double-stranded consensus sequences to reduce false positive errors. Using DS, detection of a persistent mutation utilizing defined criteria was seen in 22 (35%) patients and was strongly associated with higher rates of relapse (68% vs 13% at year 5; HR, 8.8; 95% CI, 3.2-24.5; P<0.001) and decreased survival (32% vs 82% at year 5; HR, 5.6; 95% CI, 2.3-13.8; P<0.001). MRD as defined by DS strongly outperformed MFC, which was observed in 10 (16%) patients and marginally associated with higher rates of relapse (50% vs 30% at year 5; HR, 2.4; 95% CI, 0.9-6.7; P=0.087) and decreased survival (40% vs 68% at year 5; HR, 2.5; 95% CI, 1.0-6.3; P=0.059). Furthermore, the prognostic significance of DS MRD status at the time of remission was similar on both randomized arms of the trial, predicting S0106 clinical trial outcomes. These findings suggest that DS is a powerful tool that could be used in patient management and for early treatment assessment in clinical trials.
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Mutations of ABL1 are the dominant mechanism of relapse in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). We performed highly accurate Duplex Sequencing of exons 4-10 of ABL1 on bone marrow or peripheral blood samples from 63 adult patients with previously untreated Ph + ALL who received induction with intensive chemotherapy plus a BCR-ABL1 TKI. We identified ABL1 mutations prior to BCR-ABL1 TKI exposure in 78% of patients. However, these mutations were generally present at extremely low levels (median variant allelic frequency 0.008% [range, 0.004%-3.71%] and did not clonally expand and lead to relapse in any patient, even when the pretreatment mutation was known to confer resistance to the TKI received. In relapse samples harboring a TKI-resistant ABL1 mutation, the corresponding mutation could not be detected pretreatment, despite validated sequencing sensitivity of Duplex Sequencing down to 0.005%. In samples under the selective pressure of ongoing TKI therapy, we detected low-level, emerging resistance mutations up to 5 months prior to relapse. These findings suggest that pretreatment ABL1 mutation assessment should not guide upfront TKI selection in Ph + ALL, although serial testing while on TKI therapy may allow for early detection of clinically actionable resistant clones.
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Resistencia a Antineoplásicos , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-abl/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Proteínas de Fusión bcr-abl/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación/efectos de los fármacos , Cromosoma Filadelfia/efectos de los fármacos , Proteínas Proto-Oncogénicas c-abl/química , Adulto JovenRESUMEN
Optogenetics is a technique used in various animal models and holds a potential for therapeutic possibilities in mammals. There are technical issues with the use of light sensitive ion channels: reproducible effects over time, controlling where the non-native proteins are targeted within the cell and changes in the biophysical properties of the cells they are expressed in. We used a variant of channel rhodopsin (ChR2-XXL) and targeted expression in neurons of larval Drosophila to investigate the acute and chronic activation, with light pulses, of the channels on synaptic function. The rhodopsin channel modifier all trans retinal (ATR) also plays a role in the sensitivity of the channel to light. Periods of acute, repetitive, and pulsatile blue light exposure over larval development produced attenuated responses. These blue light sensitive ion channels, with ATR, show accommodation and produce an electrical refractory period in inducing synaptic responses. The biological significance and aim of this study is to demonstrate that in controlling particular neurons or neuronal circuits with optogenetics, over time and throughout development, one will have to understand the dynamic nature of activating and silencing the light sensitive channels as well as the biophysical effects on neuronal activity.
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Conducta Animal/fisiología , Canales Iónicos/genética , Luz , Rodopsina/genética , Animales , Animales Modificados Genéticamente , Drosophila , Canales Iónicos/fisiología , Larva , Estudios Longitudinales , Neuronas/fisiología , Optogenética/métodosRESUMEN
To examine the impact of common somatic mutations in prostate cancer (PCa) on androgen receptor (AR) signaling, mouse models were designed to perturb sequentially the AR, ETV1, and PTEN pathways. Mice with "humanized" AR (hAR) alleles that modified AR transcriptional strength by varying polyglutamine tract (Q-tract) length were crossed with mice expressing a prostate-specific, AR-responsive ETV1 transgene (ETV1(Tg)). While hAR allele did not grossly affect ETV1-induced neoplasia, ETV1 strongly antagonized global AR regulation and repressed critical androgen-induced differentiation and tumor suppressor genes, such as Nkx3-1 and Hoxb13. When Pten was varied to determine its impact on disease progression, mice lacking one Pten allele (Pten(+/-) ) developed more frequent prostatic intraepithelial neoplasia (PIN). Yet, only those with the ETV1 transgene progressed to invasive adenocarcinoma. Furthermore, progression was more frequent with the short Q-tract (stronger) AR, suggesting that the AR, ETV1, and PTEN pathways cooperate in aggressive disease. On the Pten(+/-) background, ETV1 had markedly less effect on AR target genes. However, a strong inflammatory gene expression signature, notably upregulation of Cxcl16, was induced by ETV1. Comparison of mouse and human patient data stratified by the presence of E26 transformation-specific ETS fusion genes highlighted additional factors, some not previously associated with prostate cancer but for which targeted therapies are in development for other diseases. In sum, concerted use of these mouse models illuminates the complex interplay of AR, ETV1, and PTEN pathways in pre-cancerous neoplasia and early tumorigenesis, disease stages difficult to analyze in man.
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Adenocarcinoma/genética , Proteínas de Unión al ADN/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Factores de Transcripción/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Transducción GenéticaRESUMEN
Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA.
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Fibras Musculares de Contracción Lenta/metabolismo , Trastornos Musculares Atróficos/metabolismo , Péptidos/metabolismo , Receptores Androgénicos/metabolismo , Sumoilación , Transcripción Genética , Animales , Ratones , Ratones Transgénicos , Fibras Musculares de Contracción Lenta/patología , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/patología , Células PC12 , Péptidos/genética , Ratas , Receptores Androgénicos/genéticaRESUMEN
Comprehensive sequencing of tumor suppressor genes to evaluate inherited predisposition to cancer yields many individually rare missense alleles of unknown functional and clinical consequence. To address this problem for CHEK2 missense alleles, we developed a yeast-based assay to assess in vivo CHEK2-mediated response to DNA damage. Of 25 germline CHEK2 missense alleles detected in familial breast cancer patients, 12 alleles had complete loss of DNA damage response, 8 had partial loss and 5 exhibited a DNA damage response equivalent to that mediated by wild-type CHEK2. Variants exhibiting reduced response to DNA damage were found in all domains of the CHEK2 protein. Assay results were in agreement with epidemiologic assessments of breast cancer risk for those variants sufficiently common for case-control studies to have been undertaken. Assay results were largely concordant with consensus predictions of in silico tools, particularly for damaging alleles in the kinase domain. However, of the 25 variants, 6 were not consistently classifiable by in silico tools. An in vivo assay of cellular response to DNA damage by mutant CHEK2 alleles may complement and extend epidemiologic and genetic assessment of their clinical consequences.
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Neoplasias de la Mama/genética , Daño del ADN , Mutación Missense , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Estudios de Casos y Controles , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Salud de la Familia , Femenino , Prueba de Complementación Genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
In communities with high rates of consanguinity and consequently high prevalence of recessive phenotypes, homozygosity mapping with SNP arrays is an effective approach for gene discovery. In 20 Palestinian kindreds with prelingual nonsyndromic hearing loss, we generated homozygosity profiles reflecting linkage to the phenotype. Family sizes ranged from small nuclear families with two affected children, one unaffected sibling, and parents to multigenerational kindreds with 12 affected relatives. By including unaffected parents and siblings and screening 250 K SNP arrays, even small nuclear families yielded informative profiles. In 14 families, we identified the allele responsible for hearing loss by screening a single candidate gene in the longest homozygous region. Novel alleles included missense, nonsense, and splice site mutations of CDH23, MYO7A, MYO15A, OTOF, PJVK, Pendrin/SLC26A4, TECTA, TMHS, and TMPRSS3, and a large genomic deletion of Otoancorin (OTOA). All point mutations were rare in the Palestinian population (zero carriers in 288 unrelated controls); the carrier frequency of the OTOA genomic deletion was 1%. In six families, we identified five genomic regions likely to harbor novel genes for human hearing loss on chromosomes 1p13.3 (DFNB82), 9p23-p21.2/p13.3-q21.13 (DFNB83), 12q14.3-q21.2 (DFNB84; two families), 14q23.1-q31.1, and 17p12-q11.2 (DFNB85).
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Estudios de Asociación Genética , Pérdida Auditiva/genética , Homocigoto , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple/genética , Árabes , Mapeo Cromosómico , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 9/genética , Consanguinidad , Femenino , Humanos , Masculino , Linaje , FenotipoRESUMEN
Acquired platinum resistance is a serious problem in the treatment of ovarian carcinomas. However, the mechanism of the drug resistance has not been elucidated. Here, we show functional significance of restoration of BRCA2 protein by secondary BRCA2 mutations in acquired drug resistance of BRCA2-mutated ovarian carcinoma. Three ovarian cancer cell lines (PEO1, PEO4, and PEO6) were derived from a BRCA2 mutation [5193C>G (Y1655X)] carrier with ovarian carcinoma with acquired cisplatin resistance and a secondary BRCA2 mutation [5193C>T (Y1655Y)] that canceled the inherited mutation. PEO1 was BRCA2 deficient and sensitive to cisplatin and a poly(ADP-ribose) polymerase inhibitor, AG14361, whereas PEO4 was resistant. PEO4 and PEO6, derived from ascites at the time of relapse with cisplatin resistance, had the secondary mutation and were BRCA2 proficient. In vitro cisplatin/AG14361 selection of PEO1 led to restoration of BRCA2 due to another secondary BRCA2 mutation. BRCA2 depletion sensitized BRCA2-restored PEO1 clones and PEO4 to cisplatin/AG14361. Thus, restoration of BRCA2 due to secondary BRCA2 mutation is involved in acquired drug resistance of BRCA2-mutated ovarian carcinoma.
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Proteína BRCA2/genética , Proteína BRCA2/fisiología , Carcinoma/genética , Resistencia a Antineoplásicos/genética , Neoplasias Ováricas/genética , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/metabolismo , Secuencia de Bases , Carcinoma/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cisplatino/farmacología , Cisplatino/uso terapéutico , Análisis Mutacional de ADN , Resistencia a Antineoplásicos/fisiología , Femenino , Humanos , Mutación/fisiología , Sistemas de Lectura Abierta/efectos de los fármacos , Sistemas de Lectura Abierta/genética , Neoplasias Ováricas/metabolismo , ARN Interferente Pequeño/farmacología , Células Tumorales CultivadasRESUMEN
Ovarian carcinomas with mutations in the tumour suppressor BRCA2 are particularly sensitive to platinum compounds. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast-cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 (refs 3, 4), led to five different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumour that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy.
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Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Genes BRCA2 , Mutación/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Azulenos/farmacología , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Benzodiazepinas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Inhibidores de Poli(ADP-Ribosa) PolimerasasRESUMEN
CONTEXT: Genetic testing for inherited mutations in BRCA1 and BRCA2 has become integral to the care of women with a severe family history of breast or ovarian cancer, but an unknown number of patients receive negative (ie, wild-type) results when they actually carry a pathogenic BRCA1 or BRCA2 mutation. Furthermore, other breast cancer genes generally are not evaluated. OBJECTIVE: To determine the frequency and types of undetected cancer-predisposing mutations in BRCA1, BRCA2, CHEK2, TP53, and PTEN among patients with breast cancer from high-risk families with negative (wild-type) genetic test results for BRCA1 and BRCA2. DESIGN, SETTING, AND PARTICIPANTS: Between 2002-2005, probands from 300 US families with 4 or more cases of breast or ovarian cancer but with negative (wild-type) commercial genetic test results for BRCA1 and BRCA2 were screened by multiple DNA-based and RNA-based methods to detect genomic rearrangements in BRCA1 and BRCA2 and germline mutations of all classes in CHEK2, TP53, and PTEN. MAIN OUTCOME MEASURES: Previously undetected germline mutations in BRCA1, BRCA2, CHEK2, TP53, and PTEN that predispose to breast cancer; frequencies of these mutations among families with negative genetic test results. RESULTS: Of the 300 probands, 52 (17%) carried previously undetected mutations, including 35 (12%) with genomic rearrangements of BRCA1 or BRCA2, 14 (5%) with CHEK2 mutations, and 3 (1%) with TP53 mutations. At BRCA1 and BRCA2, 22 different genomic rearrangements were found, of sizes less than 1 kb to greater than 170 kb; of these, 14 were not previously described and all were individually rare. At CHEK2, a novel 5.6-kb genomic deletion was discovered in 2 families of Czechoslovakian ancestry. This deletion was found in 8 of 631 (1.3%) patients with breast cancer and in none of 367 healthy controls in the Czech and Slovak Republics. For all rearrangements, exact genomic breakpoints were determined and diagnostic primers validated. The 3 families with TP53 mutations included cases of childhood sarcoma or brain tumors in addition to multiple cases of breast cancer. CONCLUSIONS: The mutational spectra of BRCA1 and BRCA2 include many high-penetrance, individually rare genomic rearrangements. Among patients with breast cancer and severe family histories of cancer who test negative (wild type) for BRCA1 and BRCA2, approximately 12% can be expected to carry a large genomic deletion or duplication in one of these genes, and approximately 5% can be expected to carry a mutation in CHEK2 or TP53. Effective methods for identifying these mutations should be made available to women at high risk.